Field of the Invention
The present invention relates generally to an apparatus for growing crystals. More particularly, the invention concerns an apparatus for growing diffraction quality, protein crystals by vapor diffusion techniques.
Discussion of the Invention
Crystallography is a powerful analytical tool which provides precise and detailed descriptions of the structure of molecules. Over the last thirty years, small molecule crystallography has been successfully integrated into chemical research at both the basic and industrial levels. Crystallography of macromolecules is now increasingly being viewed as the ultimate analytic tool of life scientists and is expected to play a very significant role in the biotechnology revolution.
In the past, several major technical difficulties have prohibited the routine use of macromolecular cyrstallography and its complete integration into the mainstream of biological sciences. These include problems associated with data collection from X-ray diffraction instrumentation, the computational analysis of the data, and the actual growing of the crystals. The successful introduction of are detectors for the extremely rapid collection of data has significantly reduced the data collection problems. Similarly, the enormous increase in computing power and the development of sophisticated computer graphic displays have substantially reduced the problems associated with computational analysis of the data. The remaining problem, crystallization, was initially circumvented by analyzing only those macromolecules which could be obtained in abundance and which crystallized readily. The structures of macromolecules possessing those qualities have long since been solved and today, the crystallization step remains the last major barrier to the routine structural analysis of commercially significant macromolecules.
One of the most common prior art vapor diffusion methods for growing crystals is the so-called "sitting drop" method and uses a glass plate with a plurality of depressions that act as wells for the protein drops. The plates are expensive and therefore are typically washed and siliconized for every trial. A clear plastic sandwich box is typically used for the equilibration chamber. The glass plate is placed on an inverted petri dish to elevate it above the solvent in the chamber and is then sealed with grease. The carrier contains protein drops equal in number to the depressions in the plate and each drop is equilibrated against the same solvent reservoir.
Another prior art method, sometimes called the "hanging drop" method, uses a multi-well plastic cell culture plate. This method allows a single protein drop to equilibrate against its own unique solvent, as opposed to all protein samples equilibrating against the same solvent, as is the case in the "sandwich box" method. In the "hanging drop" method, each drop is placed on a glass cover slip, which is carefully inverted and placed over a greased well to seal it. This "hanging drop" method is generally superior to the "sitting drop" method, but is quite awkward and significant problems are presented in removing the crystal from the greasy cover slip.
Still another prior art vapor diffusion method for growing crystals is the so-called "sandwich drop" method. In accordance with this method, the equilibrating solution is contained within a suitable reservoir and the protein drop is sandwiched between two glass plates which are sealed in position within the apparatus using vacuum diffusion pump oil. The technique of forming the sandwiched drop with parallel flat surfaces perpendicular to the light path provides improved microscopic viewing. However, this method is costly, somewhat tedious and the necessity of using a sealing medium such as pump oil provides an undesirable source of possible contamination. Further, the requirement for sealing two glass plates substantially increases the risk of vapor leaks causing the protein drops to dry out.
The aforementioned prior art, manual methods clearly illustrate the fact that, to date, the crystallization of proteins has been largely an empirical practice. The probability of success of the particular prior art method is often proportional to the number of trial samples composed. Frequently, the total number of variables such as concentrations of protein, salt, and inhibitors along with physical parameters, such as temperature, require many thousands of attempts in order to properly sample all the possibilities and combinations. Success, if it is achieved at all, sometimes demands a nearly intolerable amount of technical application and hundreds of hours of tedious human effort.
To obviate the labor intensive requirements of the prior art methods, attention in the field has turned to automation of the crystallization trial procedure. Systems for this purpose, for both dispensing the trial solutions and their later analysis for the presence of crystals, have been designed using a variety of robotic and optical scanning procedures However, for various reasons, the prior art methods do not easily lend themselves to automation. For example, robots have substantial difficulty in manipulating small glass components and properly sealing containers with sealing mediums such as oil and grease. Further, the prior art apparatus is generally not compatible with automatic pipetting equipment, or with automated optical systems used for data collection. Additionally, because of the basic design of the prior art apparatus, the kinetics of the equilibrium process are often difficult to control and reproduce.
The apparatus of the present invention overcomes many of the drawbacks of the prior art methods and devices by providing novel crystal growing apparatus which is inexpensive, easy to use and readily compatible with automatic pipetting, sample preparation and diffraction analysis equipment. Because of the unique design of the apparatus, laboratory technicians with no particular skills, or specialized training, can use the apparatus to grow high quality crystals in very large numbers for numerous uses, including the diffraction analysis of macromolecular structures.